Adeno-associated virus (AAV) is a replication-deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5xe2x80x2 and 3xe2x80x2 ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene [B. J. Carter, in xe2x80x9cHandbook of Parvovirusesxe2x80x9d, ed., P. Tijsser, CRC Press, pp. 155-168 (1990)]. It has been shown that the ITRs represent the minimal sequence required for replication, rescue, packaging, and integration of the AAV genome.
When this nonpathogenic human virus infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a lytic helper virus, such as adenovirus (Ad) or herpesvirus. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced. The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or xe2x88x92 ss DNA genomes following cell lysis.
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA (i.e., a transgene) to cells, and various groups have studied the potential use of AAV in the treatment of disease states. As used in this application, the term xe2x80x9ctransgenexe2x80x9d means the DNA desired to be delivered to an animal, the DNA being non-AAV DNA. However, progress towards establishing AAV as a transducing vector for the delivery of DNA in the form of a desired transgene has been slow for a variety of reasons.
One obstacle to the use of AAV for delivery of DNA has been lack of highly efficient schemes for encapsidation of recombinant genomes and production of infectious virions. See, R. Kotin, Hum. Gene Ther., 5:793-801 (1994). One method which addresses this problem involves transfecting a recombinant AAV (rAAV) (which has the DNA to be delivered, but lacks rep and cap genes) into host cells followed by co-infection with wild-type (wt) AAV (which supplies the rep and cap genes) and adenovirus (which supplies at least the four adenovirus genes: E1, E2, E4 and VAI, which have been stated to be necessary for rAAV production) [see, e.g., Carter, cited above]. However, in this method co-infection is mandatory and leads to unacceptably high levels of wt AAV resulting from non-homologous recombination and contamination of the rAAV produced with wt AAV. The contamination with other viruses or plasmids demands purification of rAAV. Incubation of cells with rAAV in the absence of contaminating wt AAV or helper adenovirus yields little recombinant gene expression.
A widely recognized means for manufacturing transducing AAV virions for gene therapy entails co-transfection with two different, complementing plasmids. One of these plasmids contains a therapeutic or reporter transgene sandwiched between the two cis acting AAV ITRs. The AAV components that are needed for rescue and subsequent packaging of progeny recombinant genome are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins. In this system, the Ad helper functions are provided by a wt adenovirus or by replication-defective adenovirus with the missing E1 gene supplied by HEK 293 cells. Other variants of this method have been described. See, for example, U.S. Pat. No. 5,658,785, which refers to a mammalian host cell stably transfected with a rAAV genome and with AAV rep and cap genes, and a method for producing rAAV by infecting this host cell with a helper virus.
U.S. Pat. No. 5,658,776 refers to packaging systems and processes for packaging AAV vectors in which the AAV p5 promoter is replaced with a heterologous promoter. Alternatively, U.S. Pat. No. 5,622,856 refers to constructs and methods for AAV vector production in which the homologous p5 promoter is moved to a position 3xe2x80x2 of the rep genes, optionally flanking the rep/cap genes and repositioned p5 promoter with FRT sequences.
There remains a need in the art for additional compositions and methods permitting the efficient production of AAV and recombinant AAV viruses for use as vectors for somatic gene therapy without the inefficiency, contamination and purification problems present in the methods previously described.
The present invention allows for the efficient production of rAAV containing a desired transgene DNA. Particularly, the present invention provides both compositions and methods which enable the production of a rAAV without producing contaminating re-assembled wt AAV during rAAV production.
In one aspect, the invention provides a replication-competent hybrid adenovirus/AAV virus containing a recombinant adeno-associated viral (rAAV) vector and sufficient adenoviral sequences to permit replication of said hybrid virus in a selected host cell. In one embodiment, the hybrid virus contains a functional deletion in the wild-type adenoviral E3 region and/or a deletion of non-essential adenoviral sequences in the E4 region, such that the hybrid virus contains the sequences required for E4 ORF6 function.
In another aspect, the invention provides an adenovirus/AAV hybrid virus containing (a) adenovirus 5xe2x80x2 cis-elements necessary for replication and packaging; (b) a recombinant adeno-associated viral (rAAV) vector; (c) a deletion of adenoviral sequences from the E3 region; (d) nucleic acid sequences encoding adenovirus E1a and adenovirus E1b under the control of regulatory sequences directing expression of the E1a and E1b gene products, wherein said E1a and E1b nucleic acid sequences are located in the site of the E3 region and; (e) adenovirus 3xe2x80x2 cis-elements necessary for replication and packaging. In one suitable embodiment, the hybrid virus provides all sequences necessary to provide helper function for packaging the rAAV.
In another aspect, a method for producing recombinant adeno-associated virus (rAAV) in the absence of contaminating helper virus or wild-type virus, comprising the step of culturing a host cell comprising a replication-competent rAd/AAV and an AAV rep sequence and an AAV cap sequence under the control of regulatory sequences directing expression thereof. Suitably, the method further involves the step of controlling replication of the rAd/AAV hybrid following infection, thereby enhancing production of rAAV. In a preferred embodiment, the method of the invention simplifies purification steps because the replication-competent rAd/AAV provides the rAAV construct to be packaged and all necessary adenoviral sequences, there is no helper virus used and there is insufficient adenovirus sequence in the host cell to permit homologous recombination to a contaminating wt virus.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof